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Title: Flagellum biogenesis at the old cell division pole of the pathogenic bacterium Vibrio cholerae
Author: Green, J. C. D.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2008
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This study investigates the poorly characterised V. cholerae flagellar genes flhF and flhG and their role in flagellum localisation and biogenesis. To assess the function of flhF, which encodes the flagellar signal recognition particle (SRP) – family GTPase FlhF, a ΔflhF mutant was constructed and characterised. Deletion of flhF caused cells to be either aflagellate or have a randomly localised flagellum. Cells over-expressing flhF frequently produced two flagella at one or both poles. FlhF variants with single-residue substitutions in conserved GTPase motifs were unable to restore motility to the ΔflhF mutant indicating that these motifs were required for FlhF function. The function of FlhG, encoded by a gene located immediately 3’ of flhF, was also investigated.  A ΔflhG mutant was generated and mutant cells were found to construct between eight and ten flagella, either at the old cell pole or occasionally at both poles.  Wild type cells over-expressing flhG were non-motile and did not produce any flagella. These data indicate that FlhG may act as a negative regulator of flagellum biogenesis. FlhG, a putative member of the ParA family of ATPases contains a deviant Walker A box. FlhG variants with single-residue substitutions in the Walker box were unable to restore motility to the ΔflhG mutant, suggesting that the ATPase motif is required for function. One of the FlhG variants, FlhGK54R, had a dominant negative effect on wild type motility, suggesting that this mutant is enhanced in its ability to inhibit flagellum biogenesis. Bacterial two-hybrid analysis and pull-down assays revealed that FlhG interacted with FlrA, the master regulator of flagellar gene transcription. Several of the FlhG Walker box variants were unable to bind FlrA, indicating that ATP binding and/or hydrolysis might be central to the FlhG-FlrA interaction.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available